Lung cancer is one of the most commonly fatal human tumors. Many genetic alterations associated with the development and progression of lung cancer have been reported. Although genetic changes can aid prognostic efforts and predictions of metastatic risk or response to certain treatments, information about a single or a limited number of molecular markers generally fails to provide satisfactory results for clinical diagnosis of non-small cell lung cancer (NSCLC) (Mitsudomi et al., Clin Cancer Res 6: 4055-63 (2000); Niklinski et al., Lung Cancer. 34 Suppl 2: S53-8 (2001); Watine, BMJ 320: 379-80 (2000)). NSCLC is by far the most common form, accounting for nearly 80% of lung tumors (Society, A. C. Cancer Facts and Figures 2001 (2001)). The overall 10-year survival rate remains as low as 10% despite recent advances in multi-modality therapy, because the majority of NSCLCs are not diagnosed until advanced stages (Fry, W. A. et al., Cancer 86: 1867-76 (1999)). Although chemotherapy regimens based on platinum are considered the reference standards for treatment of NSCLC, those drugs are able to extend survival of patients with advanced NSCLC only about six weeks (Non-small Cell Lung Cancer Collaborative Group, BMJ. 311: 899-909 (1995)). Numerous targeted therapies are being investigated for this disease, including tyrosine kinase inhibitors, but so far promising results have been achieved in only a limited number of patients and some recipients suffer severe adverse reactions (Kris M. N. R., Herbst R. S. Proc. Am. Soc. Clin. Oncol. 21: 292a (A1166) (2002)).
Many genetic alterations associated with development and progression of lung cancer have been reported, but the precise molecular mechanisms remain unclear (Sozzi, G. Eur. J Cancer 37: 63-73 (2001)). Over the last decade newly developed cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC; however, each of the new regimens can provide only modest survival benefits compared with cisplatin-based therapies (Schiller, J. H. et al., N. Engl. J. Med. 346: 92-98 (2002); Kelly, K. et al., J. Clin. Oncol. 19: 3210-3218 (2001)). Hence, new therapeutic strategies, such as development of molecular-targeted agents, are eagerly awaited by clinicians.
Systematic analysis of expression levels of thousands of genes on cDNA microarrays is an effective approach to identifying unknown molecules involved in pathways of carcinogenesis (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003); Kakiuchi, S. et al., Mol. Cancer. Res. 1: 485-499 (2003); Zembutsu, H. et al., Int. J. Oncol. 23: 29-39 (2003); Suzuki, C. et al., Cancer Res. 63: 7038-7041 (2003)) and can reveal candidate targets for development of novel anti-cancer drugs and tumor markers. To isolate novel molecular targets for diagnosis, treatment and prevention of NSCLC, pure populations of tumor cells were prepared from 37 cancer tissues by laser-capture microdissection and genome-wide expression profiles of NSCLC cells were analyzed on a cDNA microarray containing 23,040 genes (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003)). In the course of those experiments, KOC1 (GenBank Accession No. NM—006547) and neuromedin U (NMU; GenBank Accession No. NM—006681) were identified as genes that were frequently over-expressed in lung tumors and indispensable for growth of NSCLC cells.
Cell-to-cell communication is a prerequisite for development and maintenance of multicellular organisms. Several intercellular information-exchange systems such as chemical synapses, gap junctions, and plasmadesmata in plant cells have long been observed, but a new transporting system involving a highly sensitive nanotubular structure, tunneling nanotubes (TNTs) between the cells, was only recently reported in mammalian cells (Rustom, A. et al., Science 303, 1007-1010 (2004). Such a structure would facilitate the selective transfer of membrane vesicles and organelles; therefore TNTs in mammalian somatic cells might contribute to a cell-to-cell transporting system(s) by carrying transcription factors or ribonucleoparticles (RNPs), as in plants (Nakajima, K. et al., Nature 413, 307-311 (2001); Lucas, W. J. et al., Nat. Rev. Mol. Cell. Biol. 2, 849-857 (2001)). Some investigators have documented interactions between some RNA-binding proteins and motor proteins like kinesin and dynein within mammalian somatic cells, as well as intercellular mRNA transport in mammalian germ cells (Brendza, R. P. et al., Science 289, 2120-2122 (2000); Chemathukuzhi, V. et al., Proc. Natl. Acad. Sci. USA 100, 15566-15571 (2003); Villace, P. et al., Nucleic Acids Res. 32, 2411-2420 (2004); Morales, C. R. et al. Dev. Biol. 246, 480-494 (2002).). However, no report has emerged describing an intercellular mRNA transporting system in mammalian somatic cells involving a complex of RNA-binding proteins and motor proteins.
The phenomenon of mRNA localization has been reported in oocytes and developing embryos of Drosophila and Xenopus and in somatic cells such as fibroblasts and neurons (King, M. L. et al., Bioessays 21: 546-557 (1999); Mowry, K. L., Cote, C. A. FASEB J. 13: 435-445 (1999); Lasko, P. J. Cell Biol. 150: F51-56 (2000); Steward, O. Neuron 18: 9-12 (1997)). Beta actin (ACTB) mRNA is localized at the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J. B., Singer, R. H. Cell 45: 407-415 (1986)) and at the growth cone of developing neurons (Bassell, G. J. et al., J. Neurosci. 18: 251-265 (1998)). The localization of ACTB mRNA is dependent on the zipcode, a cis-acting element located in the 3′ UTR of the mRNA (Kislauskis, E. H. et al., J. Cell Biol. 123:165-172 (1993)). The trans-acting factor, zipcode binding protein 1 (ZBP1), was affinity purified with the zipcode of ACTB mRNA (Ross, A. F. et al., Mol. Cell. Biol. 17, 2158-2165 (1997)). After the identification of ZBP1, additional homologues were identified in a wide range of organisms including Xenopus, Drosophila, human, and mouse (Mueller-Pillasch, F. et al., Oncogene 14: 2729-2733 (1997); Deshler, J. O. et al., Science 276: 1128-1131 (1997); Doyle, G. A. et al., Nucleic Acids Res. 26: 5036-5044 (1998)). ZBP1 family members are expressed in germ embryonic fibroblasts and in several types of cancer (Mueller-Pillasch, F. et al., Oncogene 14: 2729-2733 (1997); Mueller, F. et al., Br. J. Cancer 88; 699-701 (2003)). ZBP1-like proteins contain two RNA-recognition motifs (RRMs) at the NH2-terminal part of the protein and four hnRNP K homology (KH) domains at the COOH-terminal end.
KOC1 (alias IGF-II mRNA-binding protein 3: IMP-3) is one of the IMPs (IMP-1, IMP-2, and IMP-3), which belong to the ZBP1 family members and exhibit multiple attachments to IGF-II leader 3 mRNA and the reciprocally imprinted H19 RNA (Mueller-Pillasch, F. et al., Oncogene 14: 2729-2733 (1997)). Although KOC1 was initially reported to be over-expressed in pancreatic cancer (Mueller-Pillasch, F. et al., Oncogene 14: 2729-2733 (1997); Mueller, F. et al., Br. J. Cancer 88: 699-701 (2003)), its precise function in cancer cells or even in normal mammalian somatic cells remains unclear.
KOC1 is orthologous to the Xenopus Vg1 RNA-binding protein (Vg1RBP/Vera), which mediates the localization of Vg1 mRNA to the vegetal pole of the oocyte during oocyte maturation, and IMP-1 is orthologous to the ZBP1. IMP is mainly located at the cytoplasm and its cellular distribution ranges from a distinct concentration in perinuclear regions and lamellipodia to a completely delocalized pattern. H19 RNA co-localized with IMP, and removal of the high-affinity attachment site led to delocalization of the truncated RNA (Runge, S. et al., J. Biol. Chem. 275: 29562-29569 (2000)), suggesting that IMPs are involved in cytoplasmic trafficking of RNA. IMP-1 was able to associate with microtubles (Nielsen, F. C. et al., J. Cell Sci. 115: 2087-2097 (2002); Havin, L. et al., Genes Dev. 12: 1593-1598 (1998)), and is likely to involve a motor protein such as kinesin, myosin, and dyenin. On the other hand, Oskar mRNA localization to the posterior pole requires Kinesin I (Palacios, I. M., St. Johnston D. Development 129: 5473-5485 (2002); Brendza, R. P. et al., Science 289: 2120-2102 (2000)).
KIF11 (alias EG5) is a member of kinesin family, and plays a role in establishing and/or determining the stability of specific microtuble arrays that form during cell division. This role may encompass the ability of KIF11 to influence the distribution of other protein components associated with cell division (Whitehead, C. M., Rattner, J. B. J. Cell Sci. 111: 2551-2561 (1998); Mayer, T. U. et al., Science 286: 971-974 (1999)).
NMU is a neuropeptide that was first isolated from porcine spinal cord. It has potent activity on smooth muscles (Minamino, N. et al., Biochem. Biophys. Res. Commun. 130: 1078-1085 (1985); Domin, J. et al., Biochem. Biophys. Res. Commun. 140: 1127-1134 (1986); Conlon, J. M. et al., J. Neurochem. 51: 988-991 (1988); Minamino, N. et al., Biochem. Biophys. Res. Commun. 156: 355-360 (1988); Domin, J. et al., J. Biol. Chem. 264: 20881-20885 (1989), O'Harte, F. et al., Peptides 12: 809-812 (1991); Kage, R. et al., Regul. Pept. 33: 191-198 (1991); Austin, C. et al., J. Mol. Endocrinol. 12: 257-263 (1994); Fujii, R. et al., J. Biol. Chem. 275: 21068-21074 (2000)), and in mammalian species NMU is distributed predominantly in the gastrointestinal tract and central nervous system (Howard, A. D. et al., Nature 406: 70-74 (2000); Funes, S. et al., Peptides 23: 1607-1615 (2002)). Peripheral activities of NMU include stimulation of smooth muscle, elevation of blood pressure, alternation of ion transport in the gut, and regulation of feeding (Minamino, N. et al., Biochem. Biophys. Res. Commun. 130: 1078-1085 (1985)); however, the role of NMU during carcinogenesis has not been addressed. Neuropeptides function peripherally as paracrine and autocrine factors to regulate diverse physiologic processes and act as neurotransmitters or neuromodulators in the nervous system. In general, receptors that mediate signaling by binding neuropeptides are members of the superfamily of G protein-coupled receptors (GPCRs) having seven transmembrane-spanning domains. Two known receptors for NMU, NMU1R and NMU2R, show a high degree of homology to other neuropeptide receptors such as GHSR and NTSR1, for which the corresponding known ligands are Ghrelin (GHRL) and neurotensin (NTS), respectively. NMU1R (FM3/GPR66) and NMU2R (FM4) have seven predicted alpha-helical transmembrane domains containing highly conserved motifs, as do other members of the rhodopsin GPCR family (Fujii, R. et al., J. Biol. Chem. 275: 21068-21074 (2000); Howard, A. D. et al., Nature 406: 70-74 (2000); Funes, S. et al., Peptides 23: 1607-1615 (2002)).
A C-terminal asparaginamide structure and the C-terminal hepatapeptide core of NMU protein are essential for its contractile activity in smooth-muscle cells (Westfall, T. D. et al., J. Pharmacol. Exp. Ther 301: 987-992 (2002); Austin, C. J. Mol. Endocrinol. 14: 157-169 (1995)). Recent studies have contributed evidence that NMU acts at the hypothalamic level to inhibit food intake; therefore this protein might be a physiological regulator of feeding and body weight (Howard, A. D. et al., Nature 406: 70-74 (2000); Maggi, C. A. et al., Br J. Pharmacol. 99: 186-188 (1990); Wren, A. M. et al., Endocrinology 143: 227-234 (2002); Ivanov, T. R. et al., Endocrinology 143: 3813-3821 (2002)). However, so far no reports have suggested involvement of NMU over-expression in carcinogenesis.
Studies designed to reveal mechanisms of carcinogenesis have already facilitated identification of molecular targets for anti-tumor agents. For example, inhibitors of farnesyltransferase (FTIs) which were originally developed to inhibit the growth-signaling pathway related to Ras, whose activation depends on posttranslational farnesylation, has been effective in treating Ras-dependent tumors in animal models (He et al., Cell 99:335-45 (1999)). Clinical trials on human using a combination or anti-cancer drugs and anti-HER2 monoclonal antibody, trastuzumab, have been conducted to antagonize the proto-oncogene receptor HER2/neu; and have been achieving improved clinical response and overall survival of breast-cancer patients (Lin et al., Cancer Res. 61:6345-9 (2001)). A tyrosine kinase inhibitor, STI-571, which selectively inactivates bcr-abl fusion proteins, has been developed to treat chronic myelogenous leukemias wherein constitutive activation of bcr-abl tyrosine kinase plays a crucial role in the transformation of leukocytes. Agents of these kinds are designed to suppress oncogenic activity of specific gene products (Fujita et al., Cancer Res. 61:7722-6 (2001)). Therefore, gene products commonly up-regulated in cancerous cells may serve as potential targets for developing novel anti-cancer agents.
It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs) recognize epitope peptides derived from tumor-associated antigens (TAAs) presented on MHC Class I molecule, and lyse tumor cells. Since the discovery of MAGE family as the first example of TAAs, many other TAAs have been discovered using immunological approaches (Boon, Int. J. Cancer 54: 177-80 (1993); Boon and van der Bruggen, J. Exp. Med. 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J. Exp. Med. 178: 489-95 (1993); Kawakami et al., J. Exp. Med. 180: 347-52 (1994)). Some of the discovered TAAs are now in the stage of clinical development as targets of immunotherapy. TAAs discovered so far include MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gp100 (Kawakami et al., J. Exp. Med. 180: 347-52 (1994)), SART (Shichijo et al., J. Exp. Med. 187: 277-88 (1998)), and NY-ESO-1 (Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-8 (1997)). On the other hand, gene products which had been demonstrated to be specifically over-expressed in tumor cells, have been shown to be recognized as targets inducing cellular immune responses. Such gene products include p53 (Umano et al., Brit. J. Cancer 84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit. J. Cancer 84: 94-9 (2001)), CEA (Nukaya et al., Int. J. Cancer 80: 92-7 (1999)), and so on.
In spite of significant progress in basic and clinical research concerning TAAs (Rosenbeg et al., Nature Med. 4: 321-7 (1998); Mukherji et al., Proc. Natl. Acad. Sci. USA 92: 8078-82 (1995); Hu et al., Cancer Res. 56: 2479-83 (1996)), only limited number of candidate TAAs for the treatment of cancer are available. TAAs abundantly expressed in cancer cells, and at the same time which expression is restricted to cancer cells would be promising candidates as immunotherapeutic targets. Further, identification of new TAAs inducing potent and specific antitumor immune responses is expected to encourage clinical use of peptide vaccination strategy in various types of cancer (Boon and can der Bruggen, J. Exp. Med. 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J. Exp. Med. 178: 489-95 (1993); Kawakami et al., J. Exp. Med. 180: 347-52 (1994); Shichijo et al., J. Exp. Med. 187: 277-88 (1998); Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-8 (1997); Harris, J. Natl. Cancer Inst. 88: 1442-5 (1996); Butterfield et al., Cancer Res. 59: 3134-42 (1999); Vissers et al., Cancer Res. 59: 5554-9 (1999); van der Burg et al., J. Immunol 156: 3308-14 (1996); Tanaka et al., Cancer Res. 57: 4465-8 (1997); Fujie et al., Int. J. Cancer 80: 169-72 (1999); Kikuchi et al., Int. J. Cancer 81: 459-66 (1999); Oiso et al., Int. J. Cancer 81: 387-94 (1999)).
It has been repeatedly reported that peptide-stimulated peripheral blood mononuclear cells (PBMCs) from certain healthy donors produce significant levels of IFN-γ in response to the peptide, but rarely exert cytotoxicity against tumor cells in an HLA-A24 or -A0201 restricted manner in 51Cr-release assays (Kawano et al., Cancer Res. 60: 3550-8 (2000); Nishizaka et al., Cancer Res. 60: 4830-7 (2000); Tamura et al., Jpn. J. Cancer Res. 92: 762-7 (2001)). However, both of HLA-A24 and HLA-A0201 are one of the popular HLA alleles in Japanese, as well as Caucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al., J. Immunol. 155: 4307-12 (1995); Kubo et al., J. Immunol. 152: 3913-24 (1994); Imanishi et al., Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)). Thus, antigenic peptides of cancers presented by these HLAs may be especially useful for the treatment of cancers among Japanese and Caucasian. Further, it is known that the induction of low-affinity CTL in vitro usually results from the use of peptide at a high concentration, generating a high level of specific peptide/MHC complexes on antigen presenting cells (APCs), which will effectively activate these CTL (Alexander-Miller et al., Proc. Natl. Acad. Sci. USA 93: 4102-7 (1996)).
Although advances have been made in the development of molecular-targeting drugs for cancer therapy, the ranges of tumor types that respond as well as the effectiveness of the treatments are still very limited. Hence, it is urgent to develop new anti-cancer agents that target molecules highly specific to malignant cells and are likely to cause minimal or no adverse reactions. To achieve the goal molecules whose physiological mechanisms are well defined need to be identified. A powerful strategy toward these ends would combine screening of up-regulated genes in cancer cells on the basis of genetic information obtained on cDNA microarrays with high-throughput screening of their effect on cell growth, by inducing loss-of-function phenotypes with RNAi systems (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003)).